Continued revision and refining of a physiological pharmacokinetic model for methotrexate is being carried out and used in cooperative studies on MTX. HPLC techniques are being refined and developed so that separation and quantitation of methotrexate and its metabolites from biological fluids and tissues can be achieved. Mechanisms of action of combinations of other antitumor agents and normal metabolic body substances with methotrexate in increasing selective cytotoxic action are being investigated in the context of pharmacokinetics.